Multi-omics analysis of human mesenchymal stem cells shows cell aging that alters immunomodulatory activity through the downregulation of PD-L1

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders.

effects. Were these genes removed for the final analysis? While it's good that they were checked for dissociation gene expression patterns, removing these genes is not appropriate -the genes could also be involved in the MSC biological pathways under study.
The text references "Supplemental Table 10", but this table does not appear in the supplied Excel workbook.
In the Methods section, there are no details provided as to how the number of principal components were selected for scRNA-Seq dimensionality reduction or how the cluster resolution was determined.
Reviewer #3 (Remarks to the Author): The authors have performed an exhaustive gene and protein expression analysis of mesenchymal sroma cells (MSC). The goal of the single cell based analysis was to describe the cellular phenotype variation in MSC and compare MSC from bone marrow, adipose tissue, placenta and cord blood. The data confirmed that MSC are not homogenous in culture, and that the charachteristics of the different sources organs are maintained. Fewer proliferative and stem like cells are present in adult MSC than in perinatal MSC, while more senescence marktrers and in general G1/2 phase markers are receiprocally present. This is not new, except for the now very thorough analysis showing the shift in senescence and the cell source specific relative proportions of senescence and non-senescent cells. The data alsoe confirm many of the previous findings on factors influencing or accompanying proliferation, senescence, immuesuppressive function or differentiation. It remains unclear whether the comparison is biologically sound as the MSC from different tissue sources may as well represent different cell types, which are compared here. The transcriptomic analysis hints in this direction. The finding that PD-L1 is associated with senescence and immunesuppression was confirmed, but is not new (e.g. non referenced paper by Davies, 2017). New is the finding that GATA2, by regulating for example PD-L1, is important for maintaining a rather non-senescent phenotype and maintaining a better immune suppression in the used biolgical assays. It would be of interest whether this function can be shown in long-term cultures after multiple passageing of cells in vitro -and not only in peroxide induced senescence. The authors claim that knowing the transcriptomic profiles and factors can be used to influence senescence and thus maintain a homogenous and strong immunosuppressive phenotype in clinical products. This is a well written manuscript. However, the complex methodological procedure and amount of figures (6 Figures and 61 panels) are not needed to get the information out and should be reduced and focused on the main new findings.The specific points raised are: -elucidate the point that the MCS from different source organs are intrinsically different and cannot be compared as potentially identical cell types. Can the data be analysed under this aspect? Are 'tissue origin related differences among subpopulations...' also found between populations? -is it possible that the cell clusters just reflect different cell cycle stages, which could be misinterpreted as senecscent vs. proliferative? -donor age should be reorted (AD and BM MSC -reduce complexity of text and figues and focus on main novel findings. For example, most transcriptomic and proteomics data confirm what has been known, but novelty is limited (although the provided cell atlas is valuable).
-it should be discussed whether the detected functional differences are potentially clinically relevant (how predictive are the used suppression assays to predict clinical efficacy)?
Recommend to accept with major revision.

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We appreciate the encouraging and constructive comments from all reviewers.
Following the suggestions by the reviewers, we have performed additional experiments and provided a substantial amount of new information in the revised manuscript. Among the highlights of the new results, we have improved the analysis of our single cell RNA sequencing data by adopting more rigorous strategies, which further enhanced the credibility of our analysis results. Besides, a single-cell transcriptomic analysis of bone marrow mesenchymal stem cells (BM-MSCs) from aged and young donors was complemented and verified by exhaustive functional experiments, which provided further evidence without confounding by tissue-specific functional differences that senescence is a key determinant of the PD-L1-mediated immunosuppressive activity of MSCs. We have also provided new data to reveal that PD-L1 can be detected in extracellular vesicles (EVs) of MSCs, and cellular senescence attenuates the ability of MSCs to secrete PD-L1. Furthermore, we elucidated the mechanism by which GATA2 drives the expression of PD-L1, and by further complemented the GATA2-overexpression and -knockdown system, we provided better functional evidence for GATA2 as a critical regulating factor in restraining senescence and maintaining immunomodulatory capacity of MSCs. Finally, we reported here a clinical trial (NCT04014166) of the use of umbilical cord MSCs (UC-MSCs) in treating refractory immune thrombocytopenia (ITP). The new data are highlighted in a blue font in the revised manuscript, including the legends and labels of figures and supplementary tables. With the new results, we believe we have addressed Wbb e\ j^ [ h[l_[m[hiz YedY[hdi* ikXijWdj_Wbbo _cfhel[Z j^[ gkWb_jo WdZ mechanistic insight of our work, and broadened the impacts of our study.
A brief summary of the major new results is listed below, which are divided into five categories.
A. Adoption of more rigorous strategies to calculate DEGs, single-cell gene set score and pseudotime trajectory.

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2. Study enrollment, UC-MSC administration, participant follow-up and data analysis are shown for the three study groups with escalated therapeutic dose ( Supplementary Fig. 8c).
3. The longitudinal changes in platelet counts in the participants receiving UC-MSC treatment for 28 weeks (Supplementary Fig. 8d). =fWhj \hec j^ [i[* m[^Wl[ Yecfb[c[dj[Z _d Nkffb[c[djWho OWXb[ / j^[ X_ebe] [Zkh[ Z[iYh_fj_edi m[h[ i_cfb_\_[Z _d j^[ h[ikbj i[Yj_ed, We also present additional results to the reviewers as appendix figures at the end of our response letter (Appendix Fig. 1-6). We prefer not to include these data in the manuscript due to space limitations and the scope of our study, but we will be happy to include them if the reviewers or the editors think this necessary. Unless otherwise noted, all figure callouts below correspond to figures in the revised manuscript.   \hec WZ_fei[ j_iik[ '=@( WdZ Xed[ cWhhem '>I(* WdZ f[h_dWjWb iekhY[i X[_d] \hec fbWY [djWb Y^eh_ed_Y c[cXhWd[ 'LI( WdZ kcX_b_YWb YehZ 'P?(vYWd ceZ[b j^[ i[d[iY[dj fheY[ii WdZ Wbbem \eh _Z[dj_\_YWj_ed e\ yij[cd[iiz \WYjehi* m^_Y^j^[ Wkj^ehi _Z[dj_\_[Z Wi C=O=0 WdZ L@+ H/,L[h_dWjWb IN?i m[h[ b[ii WXb[ je Z_\\[h[dj_Wj[ j^Wd WZkbj IN?i We thank the reviewer for raising this question. We learned from the published articles that a tightly balanced core set of specific transcription factors are the major driving forces in stem cell maintenance, which are able to promote self-renewal by repressing transcription factors that initiate differentiation programs [1][2][3][4] , and aging of stem cells are known to affects their function, leading to the loss of self-renewal capacity 5 , this is why we interpreted MSCs with lower differentiation capacity as being more stemness and less senescent in the first draft. However, following the suggestion of reviewer, we have recognized that MSC stemness is a quite controversial issue even to this day. We admitted if MSCs cannot be proved to be pluripotent stem cells with self-renewal ability, it is inappropriate to use differentiation ability to illustrate their senescence status, as stronger differentiation capacity of MSCs mWi _dj [hfh[j[Z Wi yceh[ ij[cd[iiz _d cWdo published studies. In this work, our core content and main innovations are to highlight the effect of cell senescence on the expression of PD-L1 in MSCs, and to prove the regulatory effect of GATA2 on the aging process and PD-L1 expression of MSCs. The stemness and differentiation function of MSCs are not the focus of our research, but the evidence we used to verify the aging status of cells. In addition, we have provided a large amount of evidence to prove cellular senescence, such as N=++]Wb ijW_d_d] (Fig.   3e), CCK8 proliferation assay (Fig. 3c), _cckde\bkeh [iY[dY[ ijW_d_d] \eh -H2AX

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): While the authors have made a heroic effort in the revision by substantially increasing sample numbers of different ages and more functional assays to validate the role of GATA2/PD-L1 in MSC senescence, the decision to just merely delete the stemness portion not only ignores the biological identity of the studied cells, but also completely changes the hypothesis & direction of the research. In this "no-stemness " context, finding any molecular mechanism (i.e. the GATA2/PDL1 axis) loses much its significant because the only functional outcome for non-stem cell senescence, AKA 'regular' somatic cell senescence, is proliferation, because there is no issue of differentiation capacity for nonstem cells. And in this non-stem cell context, the finding of PD-L1 involvement in immunomodulation is completely expected, leaving only the modulation by GATA2 being novel. Moreover, if typical nonstem cell/somatic cell senescence is emphasized-meaning reversal and/or changes in differentiation capacity is no longer an issue-then the assessment of only proliferation is not enough, i.e. cell cycle dynamics and other more senescence-specific molecular mechanistic reversals by perturbing GATA2/PD-L1 needs to be evaluated.
The decision to just leave out stemness in the manuscript is actually problematic on several levels: 1) fundamentally, the authors are considering MSCs as stromal cells and NOT stem cells, and therefore has assigned immunomodulatory properties as a functional characteristic of M-stromal-Cs, a function which have yet to reach consensus; 2) the authors still show trilineage differentiation experiments AND use the changes in trilineage differentiation capacity as evidence of senescence (Supp . Fig 5a), so clearly the assayed cells ARE stem cells-how can these functions be just ignored?; and 3) so why do the authors still use mesenchymal STEM cells throughout the article? Perhaps a most troubling concern is that the authors are misunderstanding stem cell biology: in the reply, the statement "if MSCs cannot be proved to be pluripotent stem cells…" shows that the authors are maybe uncertain on stem cell biology in general, and pluripotent stem cells vs. somatic stem cells in particular.
The addition of clinical data perhaps demonstrates the commitment of authors to MSC translational use, but actually does not add to support the hypothesis of the manuscript itself (no involvement of GATA2, PD-L1, or senescence), so it is also unclear why this data was added in the revision.
Minor comment: The authors should make available the .rds file, including meta data, labeled assays, and reduction map information in order to ensure the reproducibility of the scRNA-seq data.
Reviewer #2 (Remarks to the Author): In their revised manuscript, Gao et al. make substantial improvements to their initial submission and respond to many of the reviewer concerns detailed in the original review. The addition of several experimental datasets further demonstrating a role for GATA2 in PD-L1 regulation, clarification of computational analyses, and important methodological details all strengthen the manuscript. However, despite these improvements, significant concerns remain regarding portions of manuscript content, particularly for single cell RNA-Seq (scRNA-Seq) analyses and interpretations.

MAJOR POINTS
• The inclusion of new mechanistic data in Figure 7 and Supplemental Figure 7 further supports a role for GATA2 in regulating senescence and PD-L1 expression. These additions significantly improve the manuscript and provide support for some of the patterns observed in scRNA-Seq data.
• There remain significant concerns regarding the degree to which cell cycle phase differences potentially contribute to the different MSC clusters defined in scRNA-Seq analysis. In the initial review, multiple reviewers raised concerns about whether differences in cell cycle phase might be driving the heterogeneity annotated as different MSC "senescent" and "non-aging" clusters. The authors helpfully address some of these points by modifying the language used in describing cell cycle assignments and reframing their interpretation of these data. However, as noted in the initial review, it still remains unclear how much of the gene expression differences in the "proliferative" vs "non-proliferative" clusters are driven simply by differences in cell cycle phase versus differences in senescence. While the authors attempt to address this in their response to initial review, the following issues remain: -The authors explain that when removing cell cycle genes from the highly variable genes used for PCA and clustering, there are minimal differences in cluster assignments (Appendix 4a-c). However, the "cell cycle genes" used are the Seurat cell cycle gene sets `cc.genes$s.genes` and `cc.genes$g2m.genes`. While very effective in calculating single cell cycle scores and regressing out these signatures, these gene sets are not comprehensive; there are many additional genes that may be associated with cell cycle that are not included in these sets. Therefore, even upon removal of these genes, remaining cell cycle-associated genes could still be driving the clustering pattern observed.
-The authors next regress out Seurat cell cycle scores and observe its impact on UMAP visualization and clustering (Appendix 4e). While they note that they still observe different clusters, it is very apparent that the clusters are far less distinct than the initial analysis, suggesting that indeed cell cycle gene expression likely contributes considerably to cluster assignment. Importantly, these critiques are not intended to suggest that the authors' interpretation of these cells as "senescent" vs. "non-aging" are necessarily incorrect (subsequent experimentation demonstrates very clearly the difference in senescent cell frequencies in these samples!); however, if the presented output of these analyses is a characterization of the transcriptomic heterogeneity of these cells, it is important to acknowledge the contributions of cell cycle to clustering. In sum, while the different clusters may represent and/or overlap with senescent vs. non-aging cells, some portion of this heterogeneity may be driven by cell cycle and/or cell cycle-associated effects.
• In initial submission, multiple reviewers commented on the potentially confounding effects of comparing MSC from different tissue sources. In the more recent submission, the authors have included a helpful comparison of BM-derived MSC from young vs old donors. Importantly, as noted in the text, all samples were collected and processed in a single technical batch, enabling direct comparisons of groups. This addition significantly strengthens the manuscript and adds additional support to the authors' claims of age-associated senescence programs. However, the manuscript also includes inter-tissue comparisons of four different tissue sources that were processed in different batches. While not necessarily a problem, direct comparisons across these samples without either 1) demonstrating the (unlikely) absence of a batch effect or 2) accounting for potential technical batch effects in differential gene expression testing could lead to spurious results. At the advice of reviewers, the authors did add pseudobulk differential gene expression testing to take advantage of their biological replication. However, in their analysis code, it appears that they did not include a design term for "batch" (which are noted and balanced across groups in Supplemental These are likely overinterpretations of these data, which while potentially supportive of the authors' model, are not really necessary to prove their points. In particular, inclusion and acknowledgment of differences in relative cell frequency per cluster (lines 252-254, Fig3b, Supplemental Fig 3a) provide much stronger data to support differences in adult vs. perinatal sources; these are controlled for tissue source and batch (Seurat integration) and should not be susceptible to gene expression differences. It is this reviewer's opinion that the same conclusions could be drawn by focusing on these frequency differences rather than the RNA velocity results.
• In response to initial review, per cell gene set scores have now been calculated and compared with AUCell, an appropriate tool for this application at single cell resolution. However, the authors also note application of AUCell to pseudobulk profiles (lines 1055-1056). It is not clear from the available analysis code how AUC scores were compared for differential pathway activity analyses in pseudobulk profiles or if this approach is appropriate.
• The clinical trial data, which while interesting in comparing MSC dosing regimens for clinical response, does not inform the manuscript's primary subject (senescence in MSCs). It seems outside the scope of the present manuscript.
• As noted in the previous review, at multiple points throughout the manuscript main text, the authors use language that suggests firm biological conclusions drawn solely from scRNA-Seq analysis. Some examples include: -Lines 148-149: "These results collectively indicate that the attenuated MSC proliferation can be ascribed to cellular senescence." -Lines 202-204: "Thus, our data at single-cell resolution indicate that the loss of proteostasis plays an important role in driving the cellular senescence of C5." -Lines 257-259: "Additionally, this heterogeneity in the dynamics of transition led to a major increase in the proportion of C7 but caused no significant expansion of C5 and C6 in adult MSCs ( Fig. 3b; Supplementary Fig. 3a)." In each of these cases, the text states firm causal relationships ("ascribed to…", "plays an important role in driving…", "led to a major increase…") inferred from the scRNA-Seq results. While the single cell gene expression data may suggest, infer, be consistent with, and/or inform future investigations, it is not sufficient to support such definitive statements, which would require validating experimentation (which is provided by the authors in several cases). These statements, which appear throughout the text, should be modified accordingly.
Reviewer #3 (Remarks to the Author): The authors have responded to the points made by the reviewers in detail and added relevant information, experimental data and explanations. The key finding that GATA2 reguates PD-L1 and thus by analogy immunosuppressive function, and that ihis is impaired on older (senescent) MSC is supported. The general correlation between ageing, stemness and differentiation properties has been more realistically described. Thus, I am of the opinion that the experimental options to strengthen the manuscript and support the finding have been exhausted.